The following discussion of the prior art is provided as technical background, to enable the features and benefits of the invention to be fully appreciated in an appropriate technical context. However, any reference to the prior art should not be taken as an express or implied admission that such art is widely known or forms part of common general knowledge in the field.
The technology for cryopreserving human and animal embryos as well as many other types of biological cells and small tissue samples is known.
In particular, during In-Vitro Fertilisation (IVF) procedures, embryo cryopreservation involves the extraction, fertilization, freezing and storing of embryos. As required, the embryos can be thawed and transferred to the uterus for normal development.
More recently, similar cryopreservation techniques have been applied to unfertilized eggs or oocytes. Oocyte cryopreservation involves the extraction, freezing and storing of the female eggs, or oocytes in an unfertilized state. As required, the eggs can be thawed, fertilized, and transferred to the uterus as embryos. The technique of freezing oocytes rather than embryos is considered desirable for medical, personal and ethical reasons.
Currently there are two known methods for cryopreserving biological cells and tissues. In order to succeed, all cryopreservation strategies, must avoid ice crystal formation, solution effects, and osmotic shock. The traditional method is to slowly cool the cells and its surrounding solution to the storage temperature and purposely initiate the formation of ice crystals remote from the cell/s. A more recent method known as vitrification, transforms the solution into a glass-like amorphous solid that is free from any crystalline structure, following extremely rapid cooling.
In both methods, it is known to use additional chemicals to avoid cell damage. These chemicals are known as cryoprotectants and may be divided into two categories, permeating and nonpermeating.
Permeating cryoprotectants are small molecules that readily permeate the membranes of cells. They form hydrogen bonds with water molecules and prevent ice crystallization. Some examples are ethylene glycol (EG), dimethyl sulphoxide (DMSO) and glycerol. At low concentrations in water, they lower the freezing temperature of the resulting mixture. However, at high enough concentrations, they inhibit the formation of the characteristic ice crystal and lead to the development of a solid, glasslike, or vitrified state in which water is solidified, but not crystalline or expanded. The toxicity at this concentration is quite high and therefore the cell can be exposed to this solution either for a very short period of time (as with vitrification techniques) or at very low temperatures, at which the metabolic rate of the cell is very low.
In contrast to the permeating cryoprotectants, nonpermeating cryoprotectants remain extracellular. Some examples are the disaccharides trehalose and sucrose. They act by drawing free water from within the cell, thus dehydrating the intracellular space. As a result, when they are used in combination with a permeating cryoprotectant, the net concentration of the permeating cryoprotectant can be increased in the intracellular space. This further assists the permeating cryoprotectant in preventing ice-crystal formation.
During vitrification, permeating cryoprotectants may be added at a high concentration while the cell's temperature is controlled at a predetermined level above freezing. However, because the toxicity of this high concentration of permeating cryoprotectant is substantial, the cell/s cannot be kept at these temperatures for long. Instead, a very short time is allowed for equilibration, after which the embryos/oocytes are plunged directly into liquid nitrogen. This extremely rapid rate of cooling not only minimizes the negative effects of the cryoprotectant on the cell, but also further protects against ice-crystal formation by encouraging vitrification.
A typical vitrification process involves exposing the cell to three or more vitrification solutions. The vitrification solutions are added to respective wells in a multi-well culture dish. The dish and solutions are warmed to predetermined temperature selected depending on the type of cell or tissue.
In a typical protocol, the cell is transferred to a first solution in a first well and washed by carefully moving the cell through the solution with a cell pipetting device. The washing process is repeated in the second, third and fourth wells for various predetermined periods of time, until the cell is ready for cryopreservation.
The cell is then drawn up with a predetermined measure of vitrification solution with a pipettor. A droplet containing the cell to be vitrified is wiped onto the hook of a fiber plug.
The fiber plug may be transferred directly into liquid nitrogen or on to the surface of a vitrification block that has been pre-cooled with liquid nitrogen. The fiber plug is placed onto the surface of the block for a minimum period during which time the cell and fluid become vitrified. The fiber plug is then inserted into a pre-chilled straw or other device located in a slot in the vitrification block before being transferred to long-term cold storage in either liquid nitrogen or liquid nitrogen vapour.
To maximize the survival chances of the cell it is very important that the process is carried out with minimal manipulation. In addition, the process and timing of washing and cooling must be adhered to with minimal variation. The process is both time consuming and requires the technician to have a relatively high level of training and skill to achieve an acceptable survival ratio.
It is an object of the present invention to overcome or substantially ameliorate one or more of the limitations of the prior art, or at least to provide a useful alternative.